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A survey of Ticks (Acari: Ixodidae) and their microbes from companion animals in Australia

Greay, Telleasha (2020) A survey of Ticks (Acari: Ixodidae) and their microbes from companion animals in Australia. PhD thesis, Murdoch University.

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Ticks are vectors of pathogens affecting companion animals and can cause tick paralysis, anaemia, dermatitis, and secondary infections. In Australia, there is currently only one known tick-borne pathogen of companion animals. Babesia canis vogeli is transmitted by Rhipicephalus sanguineus sensu lato (s.l.)1 (brown dog tick). This tick species is a potential vector of Babesia gibsoni and Anaplasma platys, which are putative tick-borne pathogens that require vector transmission studies. The lack of recognised tick-borne pathogens in Australia is likely due to the lack of research on pathogenic viruses, bacteria, and protozoa in Australian ticks.

Twenty ixodid (hard tick) species have previously been recorded on dogs, cats, and horses in Australia, including Rhi. sanguineus s.l., Ixodes holocyclus (eastern paralysis tick), and Haemaphysalis longicornis (the common name in Australia is bush tick and the common name in Asia is Asian longhorned tick), which are known and putative vectors of tick-borne pathogens. Since there have been few tick surveys in Australia since the mid-twentieth century, a nationwide survey of ixodids (Acari: Ixodidae) was conducted to identify tick species that parasitise dogs, cats, and horses. Ticks were morphologically examined to determine species, instar, and sex, and the collection locations of the different tick species were mapped using QGIS software. The companion animal owners responded to questionnaires and descriptive statistics were summarised. A total of 4,765 ticks were identified from 7/8 states and territories in Australia. Overall, 220 larvae, 805 nymphs, 1,404 males, and 2,336 females of 11 tick species were identified from 837 companion animal hosts. One novel host record was obtained for Ixodes myrmecobii, which was found on Felis catus (domestic cat) in the town of Esperance, Western Australia. The most common tick species identified included Rhi. sanguineus s.l. on dogs (73%), I. holocyclus on cats (81%), and Haem. longicornis on horses (60%). However, some ticks that were excluded from the study in Chapter 2, Subsection 2.2, could not be identified based on morphology alone. Sanger sequencing of the cytochrome c oxidase subunit 1 gene (COI) was performed to confirm their species identity. The species identified included three Ixodes trichosuri nymphs, three Haemaphysalis sp. genotype 1 and one Haemaphysalis sp. genotype 2 (potentially novel species), and Haemaphysalis lagostrophi.

Since little is known about bacteria and apicomplexan parasites in Australian ticks, genomic DNA was extracted from a subset of the ticks collected from dogs, cats, and horses (n = 711) for microbial identification. All 711 tick extracts were screened for apicomplexans at the 18S rRNA gene (18S) with conventional PCR (cnPCR) and Sanger sequencing, and n = 655 tick extracts were screened for bacteria with cnPCR and amplicon next-generation sequencing (NGS). For the amplicon NGS screening, the aim was to detect bacterial pathogens, tick-associated bacteria with unknown pathogenicity (including endosymbionts), and novel species. Therefore, the 16S rRNA gene (16S) was targeted with cnPCR for amplicon NGS. Hypervariable regions V1-2 of 16S were sequenced on the MiSeq (Illumina) platform. Reads were processed using USEARCH v10.0 and denoised into zero-radius operational taxonomic units (ZOTUs). Taxonomic assignments were made using the QIIME2 feature classifier and the Greengenes, RDP Classifier, and SILVA 16S databases, and taxonomic assignments were cross-checked against the National Center for Biotechnology Information (NCBI) non-redundant nucleotide (nr/nt) database with the BLAST® command line tool. Dominant and prevalent bacterial species included “Candidatus Midichloria spp.”, Coxiella massiliensis, Coxiella spp., and Rickettsia spp. Tick-associated and haemotropic pathogens included An. platys and “Ca. Mycoplasma haematoparvum” in Rhi. sanguineus s.l. (6.9% and 0.6% of n = 174, respectively), and Bartonella clarridgeiae and Coxiella burnetii in I. holocyclus (0.3% (1/334) for both pathogens). The prevalence of “Ca. Neoehrlichia australis” in I. holocyclus (8.4%, 28/334) was significantly higher than the prevalence of “Ca. Neoehrlichia arcana” in I. holocyclus (2.1%, 7/334) (2 = 13.3, p < 0.0005). The bacterial diversity metrics differed for tick species, ecoregions, instars, and host species, but there was a lack of statistical support for feeding status for most tick species. Inconsistencies in taxonomic assignments across Greengenes, RDP Classifier, and SILVA highlights the need for validation of taxa with more comprehensive databases such as NCBI nr/nt. Future studies on tick microbiomes that use amplicon NGS would benefit from curated and quality-checked custom-built databases. As Rickettsia species could only be identified to the genus level with 16S NGS, Rickettsia-specific NGS was used for rickettsial species identification. The citrate synthase gene (gltA) assay enabled the identification of “Ca. Rickettsia tasmanensis” in Ixodes tasmani, a co-infection of “Ca. Ri. tasmanensis” and “Ca. Rickettsia antechini” in I. tasmani, “Ca. Rickettsia jingxinensis” in Haemaphysalis spp., Rickettsia gravesii in Amblyomma triguttatum triguttatum and I. holocyclus, and four Amb. t. triguttatum were co-infected with novel Rickettsia genotypes that were most similar (97.9-99.1%) to Rickettsia raoultii and Ri. gravesii. Phylogenetic analysis of near-full length 16S of Francisella and Legionellales species obtained by Sanger sequencing of 16S confirmed that the ZOTUs identified with 16S NGS included a novel Coxiellaceae genus and species in I. tasmani, two novel Francisella species in Amb. t. triguttatum, and two novel Francisella genotypes in Haemaphysalis spp.

For the Apicomplexa screening, the aim was to determine the identity and prevalence of these organisms in the 711 tick extracts from dogs, cats, and horses. The ticks were screened for apicomplexan parasites using cnPCR and Sanger sequencing. First, a short region of the 18S rRNA gene (18S) was targeted for more sensitive cnPCR screening, then a longer region (>1 kb) of 18S was sequenced for species confirmation. The tick-borne pathogen Bab. c. vogeli was identified in two Rhi. sanguineus s.l. from dogs in the Northern Territory and Queensland (QLD). Theileria orientalis genotype Ikeda was confirmed by sequencing the major piroplasm surface protein gene p32, and was detected in three Haem. longicornis from dogs in New South Wales. Eight novel piroplasm and Hepatozoon species were identified and described and named as follows: Babesia lohae n. sp., Babesia mackerrasorum n. sp., Hepatozoon banethi n. sp., Hepatozoon ewingi n. sp., Theileria apogeana n. sp., Theileria palmeri n. sp., Theileria paparinii n. sp., and Theileria worthingtonorum n. sp. Additionally, a novel cf. Sarcocystidae gen. sp. sequence was obtained from I. tasmani, but could not be confidently identified at the genus level. An exotic tick-borne pathogen, Hepatozoon canis, was identified in I. holocyclus from a dog in QLD. The dog was located, and a blood sample was collected for Hep. canis screening. Hepatozoon canis gamonts were identified by blood smear examination, 18S sequencing, and phylogenetic analysis, which confirmed that the dog was infected with the parasite. This is the first published report of Hep. canis in Australia.

Item Type: Thesis (PhD)
Murdoch Affiliation(s): Medical, Molecular and Forensic Sciences
Supervisor(s): Ryan, Una and Magni, Paola
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